A procedure (Achilles' heel cleavage or AC) is being developed which permits cleavage of multimegabase chromosomes at unique predetermined sites; this method should become vital for efficient mapping and sequencing of large eukaryotic genomes. As an example of this method, the circular Escherichia coli genome was cut at one site (near att lambda) or at two sites (near attlambda and at the 900 kb distal site), converting it to a linear 4.7-Mb molecule or to two 0.9- and 3.8-Mb fragments, respectively. As another example, chromosome V of the lower eukaryote Saccharomyces cerevisiae was cleaved at the URA3 gene (with close to 100% efficiency) without affecting any other chromosome. The rationale is as follows: the unique cleavage site is the 20-bp lacOs operator which contains overlapping HhaI and HaeII sites (5'-AGCGCT), and is inserted at the desired sites on the DNA. The 20-bp 5'-AATTGTGAGCGCTCACAATT specificity is thus acquired by the following steps, all performed on DNA embedded in agarose microbeads: (i) methylation with M.HhaI in the presence of the LacI repressor, (ii) deproteinization followed by selective AC cleavage with HhaII, and (iii) loading of the DNA and fractionation of fragments by pulsed field gel electrophoresis (PFGE), to yield only the desired fragment(s). The objective of the project is to further refine AC and to extend its use to other genomes and other applications. It is proposed to insert many 20-bp lacOs sites into the E. coli, YACs, yeast, and Drosophila genomes at desirable sites, and (1) to map the positions of AC HaeII cuts in respect to the chromosome ends and the genetic markers, and (2) to isolate (and clone in YACs or in bacterial "genomic vectors") up to 4-Mb defined DNA fragments (for further characterization). The AC procedure will also be extended to other suitable DNA-binding proteins in conjunction with their DNA target sites that carry suitable restriction/methylation site, and will be used to identify new DNA-binding proteins while at the same time mapping and sequencing their consensus binding sites. Other AC systems will be developed which cut DNA sites specific for various transcription factors, allowing selection for function-related families of multimegabase DNA fragments. One of the novel aspects of AC is its ability to selectively isolate by PFGE only one or a few multimegabase genomic fragments, with the rest of the genome being retained in the well, unable to enter the gel. This capability renders the AC procedure potentially useful in the mapping and sequencing of large eukaryotic genomes, including the human genome.